The invention concerns the structure of the sample support plates for mass spectrometric analysis of organic samples ionized by matrix-assisted laser desorption.
The invention consists of a highly flat plate, electrically conductive at least on its surface, rigidly bonded to a base structure in such a way that together they form a body having the external dimensions of a microtitration plate, but such that thermal distortions of the surface cannot occur without bending the structure. The base structure may have both depressions for frictional gripping by a robot as well as a machine-readable identifier.
Mass spectrometry involving ionization by matrix-assisted laser desorption and ionization (MALDI) has become established as a standard procedure for the analysis of biomolecules. Time of flight mass spectrometers (TOF-MS) are used most frequently, although ion cyclotron resonance spectrometers (FT-ICR: Fourier transform ion cyclotron resonance) or high-frequency quadrupole ion trap mass spectrometers may also be used.
The biomolecules are usually located in an aqueous solution. In this context, the term biomolecules refers primarily to oligonucleotides (that is, genetic material in its various forms such as DNA or RNA) and proteins (the primary building blocks of living material), including their particular analogs and conjugates such as glycoproteins or lipoproteins.
The choice of the matrix substance for MALDI depends on the kind of biomolecule; well over a hundred different matrix substances have now become known. One of the tasks of the matrix substance is to hold the sample molecules, as well separated as possible, and to bind them to the surface of the sample support, to transfer them to the gas phase during the laser pulse by creating a vapor cloud, without destruction of the biomolecules and if possible without attachment of the matrix molecules, and finally, to ionize them through protonation or deprotonation. For this task, it has been found favorable for the analyte molecules to be incorporated in some way in the largely crystalline matrices as they crystallize on the surface of the sample support, or at least to be incorporated in the boundary surfaces between the small crystals that form during crystallization.
A range of different methods have become known for the application of the sample and the matrix. The simplest of these is application by pipetting a solution containing the sample and matrix onto a cleaned, metallic sample support. The droplet of solution creates a wetted area on the metal surface, whose size corresponds approximately to that of the diameter of the droplet and depends on how hydrophilic the metal surface is and on the properties of the droplet. After the solution has dried, a sample spot consisting of small matrix crystals is formed, having the size of the wetted area, although the quantity deposited over the wetted area is generally not evenly distributed.
For matrix substances that are either only slightly soluble in water, or not at all, such as -cyano-4-hydroxy-cinnamic acid, it has been found favorable to first create a very thin layer of crystals on the surface before applying the aqueous solution of analyte, for instance by applying a solution of the matrix substance in acetone.
An improved method of sample application has been described in German Patent No. DE 197 54 978 C1, in which the surface of the sample support is provided with extremely small, easily wetted (hydrophilic) anchor areas for the sample droplets, located on the desired grid of sample spots and surrounded by an environment that is not easily wetted (hydrophobic). The droplets containing the dissolved analyte molecules that are pipetted onto the surface attach themselves to these anchor regions and crystallize there much more evenly than they do without an anchor. The crystal conglomerates bind to the surface of the sample support in these hydrophilic anchor regions quite strongly.
A favorable method of sample application is known for oligonucleotides, but is restricted to use with silicon chips. The oligonucleotides bonded to the surface of the chips have microdroplets of matrix solution (3-HPA) containing only a few hundred picoliters shot at them by piezo-electrically driven micropipettes, resulting in a crystal structure with an evenly distributed sensitivity to the MALDI process.
All these procedures for the application and crystallization of the samples depend, however, very strongly on the properties of the surface, and in particular on the properties of the hydrophilic anchor surfaces. These properties include the chemical composition of the surface of the support, the degree to which the surface has been oxidized, and most particularly its smoothness. It is especially important that extreme surface cleanliness is achieved, because even the slightest traces of impurities can seriously affect the MALDI process. It is particularly important that no alkali ions emerge from the surface into the applied sample solution.
If time of flight mass spectrometers are used for the analysis, the surface of the sample support is also required to be extraordinarily flat. Undulations in the surface may not exceed a few tens of micrometers, otherwise the determination of the mass from the flight time is made more difficult.
Up until now it has been found that only a few types of sample support material are, at least to some degree, universally usable. These particularly include (1) smooth rolled stainless steel sheet, approximately 3 mm thick, with a highly shining surface; (2) glass plates onto which a conductive layer has been vaporized; (3) silicon wafers. The critical significance of the nature of the surface can be seen from the fact that, for instance, a milled stainless-steel surface is inferior to a rolled surface. For some types of sample, on the other hand, ground surfaces are favorable, although, once again, grinding a rolled surface is better than grinding a milled surface.
Previous experience has demonstrated that all the materials that are favorable to use have the form of more or less thin plates.
If sample support plates are to be handled automatically, it is favorable to retain the form of microtitre plates that has become an industry standard and use it for the sample support plates also. It is true that this industrial standard does not have an entirely unambiguous definition; a number of attempts at standardization have been made, and these differ from one another in details that are, however, not important here. The microtitre plate is significantly thicker than the favorable plate material described above. Only sample support plates having approximately the form of microtitre plates can be processed and handled by commercially available pipetting robots. Identifiers can be printed and read on the front face, usually a barcode. The plates can be picked up by standardized grippers, and the sample droplets can be applied to them with the aid of multi-pipette heads. They can be stacked in magazines or inserted in appropriate drawer-like storage containers. The underside of the microtitre plate functions, when they are stacked in magazines, as a relatively well sealed, at least dust-proof, cover for the plate beneath.
If the underside is made of a material that does not permit condensation of the matrix material, which continuously undergoes light evaporation, stacking the plates can permit a long storage capacity for plates that have had samples and matrix material applied to them.
The use of MALDI sample supports having the form of microtitre plates as a base onto which samples may be placed by multiple pipetting heads has already been described in German Patent No. DE 196 28 178 C2 (corresponding to British Patent No. GB 2 315 329 or U.S. Pat. No. 5,770,860).
The form of a microtitre plate can only be created by using an underlying base that must be bonded to the sample plate. Removable structures are already commercially available for sample supports in the form of plates. In accordance with the requirements of Good Laboratory Practice (GLP), observed nowadays in all certified laboratories, the sample supports must be provided with a permanently attached identification. Removable base structures can not, however, be given identifications in conformity with GLP.
The finished assembly of the sample support plates consisting of the sample plate and the base structure must be suitable for use in a vacuum, and must not release polluting substances, neither in the vacuum of the mass spectrometer nor in the washing baths, that could collect on the surface of the support and thus contaminate the sample or disturb the MALDI process. It is of particular importance that the surface of the sample supports do not bend or distort under the influence of temperature changes.
Base structures for the sample support plates can economically be made from injection molded metal or, particularly economically and favorably, from a vacuum-compatible plastic. A stainless steel sample support plate, however, exhibits a thermal expansion of 12xc3x9710xe2x88x926 Kxe2x88x921. This coefficient of expansion is larger than that of any other metals or metal alloys, but significantly smaller than that of the plastics that can be used here. While there are, at the extreme, plastics with expansion rates that are this low, they do not satisfy the requirements for vacuum compatibility and washability.
As a stainless steel base structure is too expensive, and because pairs of materials with the same thermal expansion are not found, it is not possible to glue the sample plate and the base structure together, because thermal bending, as with a bimetallic strip, will always occur, which in this case would prevent their use in mass spectrometers. Adhesives, moreover, do not generally satisfy the requirement that they will not evolve gases or that impurities will not be released during washing.
Similar considerations also apply to sample support plates made of glass or silicon: here again, it is difficult to find pairs of materials with the same expansion coefficients. Known pairs that do have the same expansion coefficient, such as Kovar glass and Kovar metal, are expensive and difficult to machine.
The fundamental idea of the invention is to bond the sample plate and the base structure only at a small number of pointsxe2x80x94preferably at only three pointsxe2x80x94in such a way that while differences in the expansion of the sample plate and the base structure may indeed generate compressive or tensile forces in the plane of the plate, bending forces or twisting forces can nevertheless not be exerted on the sample plate.
This approach provides considerable freedom in relation to the selection of the material for the base structure. It is therefore possible to use an economical injection molding or an economically processed plastic that satisfies the requirements for washability and vacuum compatibility.
The sample plate and the base structure can, for instance, be rigidly joined together by means of three protrusions on the base structure pressed into holes in the sample plate, implemented in such a way that a small clearance remains between the sample plate and the base structure. Twisting or bending forces cannot be transferred through three protrusions. It is also, however, possible for metallic or non-metallic nails similar to rivets to be pressed through holes in the sample plate into recesses in the base structure. The rivet-like nails can have slightly conical heads that can only apply forces to the sample plate via a narrow contact rim, but cannot apply bending forces through full-faced pressure against the sides of the hole. The elastic or non-elastic flexibility of the rivet-like nails or the protrusions can even, to a limited extent, take up and balance shearing forces, so that only small tensile or compressive forces are generated in the sample plate, an arrangement that is particularly favorable for glass sample plates. Small raised support areas on the base structure around the connection points provide a clearance over a wide area between the sample plate and the base structure, so that twisting or bending of the base structure is not transmitted to the sample plate.
The base structure can have the form of a frame, or have the full area of a plate. Using a full plate as the base structure can, through the shape of the underside structure, in turn serve as a good cover for a sample support situated beneath it. The base structure can be provided with an inseparable barcode or with a transponder that gives the identity of the sample support in conformity with GLP. A transponder can even contain an occupancy status and a history of use. The base structure can, at its edge, also be provided with special holes or grooves to facilitate reliable gripping by robots. Grooves to assist insertion into the vacuum system of the mass spectrometer can also be provided here.
This allows the sample plate to have precisely the same area as a microtitre plate, although it can also be smaller, in which case the base structure can supply the missing edge with sufficient play. It is even possible to use divided sample plates, each having smaller dimensions. In particular, the sample plate can carry a pattern of hydrophilic anchors in a hydrophobic environment.